Neuronal activity is required for proper neural development, as well as for the maintenance of neural circuits, adaptation to changing environments, learning and memory formation. Electrical activation of neurons induces transient increases in the intracellular calcium (Ca2+) concentration, which can propagate to the nucleus thereby linking synaptic stimulation to gene regulatory events. However, it is unclear if and how different spatial-temporal patterns of activity give rise to distinct genomic responses. In this thesis I used optogenetic method to investigate activity dependent regulation of signaling pathways and gene expression in cultured hippocampal neurons. The goal was to investigate whether intrinsic activity could be mimicked in a controllable way, and if yes, how this was achieved. Specifically, I used Channel- rhodopsin (ChR2), a light-gated, cation-selective, small membrane channel isolated from Chlamydomonas reidhardtii. ChR2 can be activated with blue light and functions without any addition of cofactor when expressed in mammalian systems. I used transfection and adenoassociated virus-mediated gene transfer to express ChR2 in cultured hippocampal neurons, which afterwards could be stimulated and activated with blue LED pulses to reliably induce action potential firing and Ca2+transients. A double mutant variant of ChR2, ChR2-ab, was made, characterized and its cellular functions were studied. The results indicated that ChR2-ab inherited the properties from its previous single mutant, being more sensitive, longer opening and bi-stable. Neurons expressing ChR2-ab responded to light exposure with membrane depolarization, which ultimately led to the expression of a previously characterized pool of activity-dependent genes. ChR2-ab-mediated induction of these genes was L-type voltage-dependent Ca2+ channel dependent, as it was completely blocked with nifedifine. Similarly, treatment of ChR2-ab-expressing neurons with NMDA receptor blockers MK-801 and APV inhibited the light-evoked induction of atf3, ifi202b, inhba and serpinb2. When synaptic activity was inhibited with Na+channel blocker TTX to prevent action potential fir- ing, ChR2-ab-mediated gene induction of atf3, inhba, npas4, nr4a1 and serpinb2 was also restrained. Expression of CaMBP4, an inhibitor of nuclear Ca2+/CaM complex, revealed that ChR2-ab-mediated gene induction dependent on nuclear Ca2+signaling. Interfering the function of CREB/CBP transcription complex with E1A protein dimin- ished gene expression induced with ChR2-ab. Whole-cell patch-clamp and signaling pathway analysis revealed that neurons expressing ChR2-ab had higher basal activity, with more frequent mEPSC and higher level of pCREB and pERK. However, some ChR2-ab-expressing cells died after light application on day in vitro 14. Pharmacological analysis suggested that this was due to activation of extra-synaptic NMDA receptors during ChR2-ab activation. In an alternative strategy to induce activity, ChR2 was targeted to the membrane of endoplasmic reticulum using various targeting sequences with the goal to directly trigger Ca2+release from this internal Ca2+store. Additionally, transcription analysis with catFISH method revealed that a single nuclear Ca2+ transient induced with Bicuculline treatment was capable and sufficient to initiate transcription of arc gene.
|Supervisor:||Bading, Prof. Dr. Hilmar|
|Date of thesis defense:||10 May 2013|
|Date Deposited:||23 May 2013 09:22|
|Faculties / Institutes:||The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences|
|Subjects:||500 Natural sciences and mathematics
570 Life sciences
|Controlled Keywords:||calcium signaling, neuronal activity, channelrhodopsin|